Project Summary Most cellular processes in both the embryo and the adult are controlled by either differential expression or modification of regulatory proteins. We will develop proteomic tools to study quantitatively the effect of protein expression levels and post-translational modification on key signaling pathways in cells, and probe the complex behavior of these pathways in vertebrate embryonic development. Our premise is that highly reliable and very accurate quantitative profiling of protein levels and phosphorylation along with proper mathematical analysis will lead to insights into how signaling pathways communicate information and enable cells to make decisions on a cellular and tissue level. Phosphorylation is thought to be the major regulatory modification of proteins. At least half of all mammalian proteins are phosphorylated, yet for most phospho-sites we do not know which kinase is responsible for the phosphorylation, and hence how the target protein is regulated. We propose to dramatically advance this situation via a systems biology framework for studying phosphorylation cascades in mammalian cells in culture and in early vertebrate embryonic development, using Xenopus laevis embryos as a model system. We have shown how manipulation of kinase activity, with poly-specific kinase inhibitors, can identify the relationship between a phenotype and a specific kinase. By measuring phosphorylation sites quantitatively by MS (using each individual phosphosite as its own phenotype) we plan to provide a kinase- substrate map at a genome scale for both human and Xenopus, and use this to connect kinase activity to cellular phenotype. Our team is comprised of a close and well established collaboration between an expert embryologist/cell biologist/biochemist (Kirschner), an expert in systematic analysis and statistical inference (Peshkin) and an expert in proteomic mass spectrometry (Gygi). Our recently developed methods will allow us to reach unprecedented depth of quantification of proteins and protein post-translational modifications. In this grant we will develop further innovations in experimental design, analytical improvements and improvements in MS. The large size of Xenopus embryos permits organism-level studies of such sensitivity that they can be used on a relatively small number of experimentally manipulated embryos. Our efforts will produce systems- level knowledge of the dynamic protein states in cells and in cell populations in early vertebrate development. The resulting data sets and methods should be a powerful resource for vertebrate embryology and could be informative for heritable defects in human embryos. Many embryonic pathways, such as MAPkinase, Wnt, and hedgehog, are also very important in childhood development, tissue and cell turnover in adults, regenerative medicine, and diseases of the immune system and cancer. As many of these pathways and kinase relationships seem conserved across stages of development and across vertebrate species, there is promise of rapid transfer from model system to human patients. In particular we expect that our approach will also serve to facilitate systems-level pharmacology approaches to therapy.